Candidate: I have been a post-doctoral researcher in the lab of Prof. Margaret Goodell at Baylor College of Medicine (Houston, TX) for three years, coming from a Ph.D. at the University of Queensland (Brisbane, QLD, Australia) in May, 2006. I obtained an initial fellowship from the Australian NHMRC to fund the first two years of my post-doc, during which I have studied the molecular regulation of hematopoietic stem cell differentiation and the epigenetic mechanisms that control stem cell fate. I am now applying for more senior fellowships and beginning to take the initial steps to establishing my own research programs. The timing of this application represents a crossroads in my career, where I am beginning to transition to independence and initiate my own research programs. My work in understanding the molecular regulation of hematopoietic stem cells is motivated by a desire to improve the lives of patients afflicted with hematopoietic disorders and my ultimate career goal is to see the implementation of meaningful discoveries in HSC biology to help drive novel clinical outcomes. I believe that the work outlined in this proposal could ultimately lead to novel therapeutic approaches for a wide range of patients. Obtaining an NIH Pathway to Independence Award (K99/R00) will allow me to gain additional research training in the mentored phase of the award with activities outlined in the career development section such as seminars, journal clubs, scientific conferences, additional training in research techniques and didactic coursework as well as exposure to cutting edge techniques in stem cell biology. With additional training, I will be able to pursue an independent research position in a highly ranked academic and research environment with an emphasis in translational research during the independent phase of the award. My career goals during the mentored phase of the grant include publishing three first-author papers in high-impact journals, learning research techniques in epigenetics and computational biology and completing the career development activities to enhance my scientific background. My long-term career goals during the mentored phase of the grant include establishment of a successfully independent research program, publishing as a senior author in high-impact journals and obtaining independent funding through NIH R01's or other mechanisms. Ultimately, I would like my work to make a significant contribution to the understanding of HSC biology and its relevance to human disease states and to translate findings from my research into novel clinical treatment strategies. Environment: Baylor College of Medicine (BCM) is a premier research institute that is committed to research, unraveling the mysteries of the human body, and finding new ways to cure disease and improve health. As part of the Center for Cell and Gene Therapy (CAGT) and the Stem Cells and Regenerative Medicine (STaR) Center, I will have access to diverse core facilities with expertise in microarray, flow cytometry, microscopy and embryonic stem cells as well as access to a high-level, pathogen-free barrier utilizing ventilated racks with a limited access security system. Moreover, the diverse interests of the faculty of these Centers includes hematopoiesis, stem cell biology, hemopoietic stem cell transplantation, gene therapy, immunotherapy, and vector development and provides ample opportnity for collaboration to further my scienific training. Research: From my post-doctoral work, I have identified a project concerning the role of DNA methylation in hematopoietic stem cell (HSC) lineage fate specification. I have determined that there is differential expression of DNA methyltransferase enzymes between HSC subtypes, with myeloid-biased HSCs expressing higher levels of Dnmt3a and lymphoid-biased HSCs having higher expression of Dnmt3b, and I hypothesize that HSC lineage fate determination is at least partly controlled by the actions of DNA methyltransferases. In this study I propose to examine the functional effects of the DNA methyltransferases Dnmt3a and Dnmt3b in HSCs. The experiments proposed here will shed new light on the role of DNA methylation in HSC function and begin to uncover the epigenetic mechanisms regulating stem cell biology. There is mounting evidence for the importance of epigenetic mechanisms in the regulation of gene transcription and normal cell function, and this field of study represents the next major question in stem cell research. Moreover, there is ample evidence suggesting aberrant DNA methylation contributes in some as of yet undefined ways to various human diseases including certain hematopoietic disorders. I believe the basic research outlined in this project can contribute to the fundamental understanding of epigenetic regulation of stem cell function and has direct relevance to progression of human disease states. PUBLIC HEALTH RELEVANCE: Aberrant DNA methylation has been associated with various leukemias, however the mechanisms involved and prognostic implications of this are not understood. Treatment of myeloid leukemia patients with drugs inhibiting DNA methylation has improved clinical outcomes and DNA methylation patterns of certain tumor suppressor genes can predict the risk of relapse of acute myeloid leukemia patients in clinical remission. The experiments proposed here will shed new light on the role of DNA methylation in normal hematopoietic stem cell function which will hopefully provide insight into how abnormal DNA methylation contributes to certain leukemias.